Human myelin basic protein (HBP) isolated from whole CNS tissue or from its myelin fraction contains two HBP-related polypeptides very similar in size and charge to the 18.5 K major basic protein. In our original isolation, we assumed these to be breakdown products arising during extraction. However, a modified and improved isolation procedure (described in last year's annual report) did not change the relative amount of these polypeptides (HBP) and "fragments"). During the isolation, crude HBP is purified by ion-exchange chromatography. Fraction 1 (component 1, the last peak eluted from the column) was identified as the 18.5 K major basic protein. Fraction 3 (third from the last peak) was approximately 50% of the 18.5 K basic protein (minus 2 positive charges) and 50% other proteins which were smaller than HBP but had the same charge. Since it has been impossible to separate the 18.5 K major basic protein from the two polypeptide fragments by our present purification techniques (ion-exchange chromatography, molecular sieving and HPLC) we developed two new procedures - 1) tryptic mapping of thrombic fragments of HBP by HPLC and 2) electrophoretic transfer of peptides from 15% slab gels. By utilizing these two procedures plus amino acid analysis of the tryptic peptides, we were able to identify one of the smaller proteins in Fraction 3 as HBP minus the first 18 amino acid residues. The second small polypeptide in Fraction 3 has only been tentatively identified. These data confirm the fact that human myelin basic protein is mainly the 18.5 K major basic protein and two smaller "fragments" which we know are not products of our isolation procedure. These smaller "fragments" of HBP may be the result of autolysis in situ, and/or the result of a gene deletion.